Nucleic acids encoding anti-VEGF-A antibodies and uses thereof

ABSTRACT

The present invention relates to antibodies having activity against a vascular endothelial growth factor (VEGF), and methods of making and using such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/327,362, filed on Feb. 22, 2019, which is a U.S. NationalStage application of International Application No. PCT/EP2017/071106,filed on Aug. 22, 2017, which claims benefit under 35 U.S.C. § 119(e) ofthe U.S. Provisional Application No. 62/378,391, filed on Aug. 23, 2016.Each of the above listed applications is incorporated by referenceherein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled 106105-0512_SL.txt, createdon Sep. 10, 2020, and having a size of 117,266 bytes.

FIELD OF THE INVENTION

The invention relates to antibodies having activity against vascularendothelial growth factor (VEGF) and uses of such antibodies.

BACKGROUND TO THE INVENTION

Angiogenesis, the formation of new blood vessels from existingvasculature, is a complex biological process required for the formationand physiological functions of virtually all the organs. It is anessential element of embryogenesis, normal physiological growth, repairand pathological processes such as tumour expansion. Normally,angiogenesis is tightly regulated by the local balance of angiogenic andangiostatic factors in a multi-step process involving vessel sprouting,branching and tubule formation by endothelial cells (involving processessuch as activation of endothelial cells (ECs), vessel destabilisation,synthesis and release of degradative enzymes, EC migration, ECproliferation, EC organisation and differentiation and vesselmaturation).

In the adult, physiological angiogenesis is largely confined to woundhealing and several components of female reproductive function andembryonic development. In disease-related angiogenesis which includesany abnormal, undesirable or pathological angiogenesis, the localbalance between angiogenic and angiostatic factors is dysregulatedleading to inappropriate and/or structurally abnormal blood vesselformation. Pathological angiogenesis has been associated with diseasestates including diabetic retinopathy, psoriasis, cancer, rheumatoidarthritis, atheroma, Kaposi's sarcoma and haemangioma (Fan et al, 1995,Trends Pharmacology. Science. 16: 57-66; Folkman, 1995, Nature Medicine1: 27-31). In cancer, growth of primary and secondary tumours beyond 1-2mm3 requires angiogenesis (Folkman, J. New England Journal of Medicine1995; 33, 1757-1763).

VEGF is a potent and ubiquitous vascular growth factor. Prior toidentification of the role of VEGF as a secreted mitogen for endothelialcells, it was identified as a vascular permeability factor, highlightingVEGF's ability to control many distinct aspects of endothelial cellbehaviour, including proliferation, migration, specialization andsurvival (Ruhrberg, 2003 BioEssays 25:1052-1060). VEGF-A was the firstmember of the VEGF family of structurally related dimeric glycoproteinsbelonging to the platelet-derived growth factor superfamily to beidentified. Beside the founding member, VEGF-A, the VEGF family includesVEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) andendocrine gland-derived VEGF (EG-VEGF). Active forms of VEGF aresynthesised either as homodimers or heterodimers with other VEGF familymembers. Human VEGF-A exists in six isoforms generated by alternativesplicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. Theseisoforms differ primarily in their bioavailability, with VEGF165 beingthe predominant isoform (Podar, et al. 2005 Blood 105(4):1383-1395) butwith the others also having biological activity. The regulation ofsplicing during embryogenesis to produce stage- and tissue-specificratios of the various isoforms creates rich potential for distinct andcontext dependent behavior of endothelial cells in response to VEGF.

VEGF is believed to be an important stimulator of both normal anddisease-related angiogenesis (Jakeman, et al. 1993 Endocrinology:133,848-859; Kolch, et al. 1995 Breast Cancer Research and Treatment:36,139-155) and vascular permeability (Connolly, et al. 1989 J. Biol.Chem: 264,20017-20024). Antagonism of VEGF action by sequestration ofVEGF with antibodies can result in reduction of tumor growth (Kim, etal. 1993 Nature: 362,841-844). Heterozygous disruption of the VEGF generesulted in fatal deficiencies in vascularisation (Carmeliet, et al.1996 Nature 380:435-439; Ferrara, et al. 1996 Nature 380:439-442).

There is at least one commercially marketed anti-VEGF-A antibody, whichis Avastin®. However, there are serious, sometimes fatal, toxicitiesassociated with its use, including non-gastrointestinal fistulas,thromboembolic events, hypertension, reversible posteriorleukoencephalopathy syndrome, etc. As such, there is an unmet need atleast as it relates to improving the safety associated with targetingVEGF-A. To this end, the antibodies of the invention have bindingcharacteristics that support such an improvement over the art, includingthe ability of these antibodies to differentially bind VEGF-A isoforms.

SUMMARY OF THE INVENTION

The invention relates to binding molecules, including antibodies, thatbind to VEGF-A. The invention further relates to binding molecules,including antibodies, that bind to one or more VEGF-A isoforms withgreater affinity when compared to one or more other VEGF-A isoforms. Theinvention also relates to binding molecules, including antibodies, thatbind to VEGF-A and reduce the activity of at least one biologicalactivity of VEGF-A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Representative data demonstrating binding to human VEGF165 andmouse VEGF164.

FIG. 2 . Representative data demonstrating lack of binding to VEGF121.

FIG. 3 . Sequence alignment of clone E06 and the most sequencehomologous germline genes. Figure discloses SEQ ID NOS: 119-122,respectively, in order of appearance.

FIG. 4 . Representative data demonstrating improved binding of affinityoptimized variants.

FIG. 5 . Representative data demonstrating improved binding of affinityoptimized variants as Fabs and IgGs. Fabs are shown in the top twographs. IgGs are shown in the bottom two graphs.

FIG. 6 . Representative data demonstrating binding of affinity optimizedvariants to murine VEGF164.

FIG. 7 . Representative data demonstrating lack of binding of affinityoptimized variants to VEGF121.

FIG. 8 A-B. Representative data demonstrating activity of affinityoptimized variants in functional cell-based assays.

FIG. 9 . Representative data demonstrating activity of an affinityoptimized variant in a retinal vasculogenesis model.

DETAILED DESCRIPTION Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such can vary. As used in this specification andthe appended claims, the singular forms “a,” “an” and “the” includeplural referents unless the context clearly dictates otherwise. Theterms “a” (or “an”), as well as the terms “one or more,” and “at leastone” can be used interchangeably herein. Further it is understood thatwherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

As used herein, the term “binding molecule” refers to a molecule that iscapable of binding to a target molecule or antigen in a manner similarto that of an antibody binding to an antigen. Examples of bindingmolecules include full-length antibodies and antigen-binding fragments.Examples of “antigen-binding fragments” of an antibody include (i) a Fabfragment, a monovalent fragment that includes a VL, VH, CL and CH1domain of an antibody; (ii) a F(ab′)2 fragment, a bivalent fragment thatincludes two Fab fragments linked by a disulfide bridge at a hingeregion; (iii) a Fd fragment that includes the VH and CH1 domains; (iv) aFv fragment that includes VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which includes a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Antigen-binding fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins. In one embodiment, the antigen-binding fragmentincludes a single chain antibody, including, for example, a“single-chain variable fragment” or “scFv.” scFv refers to a fusionprotein that includes at least one variable region of a heavy chain (VH)and at least one variable region of a light chain (VL) of animmunoglobulin. These single chain antibody fragments can be obtainedusing conventional techniques known to those with skill in the art. Forexample, the VH and VL domains of a Fv fragment, which are encoded byseparate genes, can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single polypeptide chain inwhich the VH and VL regions pair to form a monovalent molecule (See,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883).

Complementarity determining regions (CDRs) are responsible for antibodybinding to its antigen. CDRs are determined by a number of methods inthe art (including Kabat (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)); Chothia (Chothia and Lesk,J. Mol. Biol. 196:901-917 (1987)); IMGT (ImMunoGeneTics) (Lefranc, M. P.et al., Dev. Comp. Immunol. 27: 55-77 (2003)); and other methods).Although specific CDR sequences are mentioned and claimed herein, theinvention also encompasses CDR sequences defined by any method known inthe art.

As use herein, the term “subject” refers to any member of the subphylumcordata, including, without limitation, humans and other primates,including non-human primates such as chimpanzees and other apes andmonkey species; farm animals such as cattle, sheep, pigs, goats andhorses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs; birds, includingdomestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like are also non-limitingexamples.

Binding Molecules

A binding molecule can include a full length or intact antibody, anantibody fragment, including an antigen binding fragment, a human,humanized, post-translationally modified, chimeric or fusion antibody,immunoconjugate, or a functional fragment thereof.

Suitable immunoglobulin molecules or portions thereof of the invention(i.e., binding molecules) can be or are derived from any isotype (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), sub-isotype (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or allotype (e.g., Gm, e.g., G1m(f, z, a or x),G2m(n), G3m(g, b, or c), Am, Em, and Km(1, 2 or 3)). Immunoglobulinmolecules can include light chains classified as either lambda chains orkappa chains based on the amino acid sequence of the light chainconstant region.

Production of Binding Molecules

Recombinant DNA methods for producing and screening for polypeptides,such as the binding molecules described herein, are known in the art(e.g. U.S. Pat. No. 4,816,567). DNA encoding the binding molecules orfragments thereof, for example, DNA encoding a VH domain, a VL domain,an scFv, or combinations thereof can be inserted into a suitableexpression vector, which can then be transfected into a suitable hostcell, such as E. coli cells, simian COS cells, Chinese Hamster Ovary(CHO) cells, or myeloma cells that do not otherwise produce an antibodyprotein, to obtain the binding molecule.

Suitable expression vectors are known in the art. An expression vectorcan contain a polynucleotide that encodes an antibody linked to apromoter. Such vectors may include the nucleotide sequence encoding theconstant region of the antibody molecule (see, e.g., U.S. Pat. Nos.5,981,216; 5,591,639; 5,658,759 and 5,122,464) and the variable domainof the antibody may be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains. The expression vector can be transferred to a host cell byconventional techniques and the transfected cells can be cultured byconventional techniques to produce the binding molecule.

Mammalian cell lines suitable as hosts for expression of recombinantantibodies are known in the art and include many immortalized cell linesavailable from the American Type Culture Collection, including but notlimit to CHO cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),human epithelial kidney 293 cells, and a number of other cell lines.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the binding molecule. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483,Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that doesnot endogenously produce any functional immunoglobulin chains), SP20,CRL7O3O and HsS78Bst cells. Human cell lines developed by immortalizinghuman lymphocytes can be used to recombinantly produce monoclonalantibodies. The human cell line PER.C6®. (Crucell, Netherlands) can beused to recombinantly produce monoclonal antibodies. Additional celllines which may be used as hosts for expression of recombinantantibodies include insect cells (e.g. Sf21/Sf9, Trichoplusia niBti-Tn5b1-4) or yeast cells (e.g. S. cerevisiae, Pichia, U.S. Pat. No.7,326,681; etc.), plants cells (U.S. 20080066200); and chicken cells(WO2008142124).

Antibodies can be stably expressed in a cell line using methods known inthe art. Stable expression can be used for long-term, high-yieldproduction of recombinant proteins. For stable expression, host cellscan be transformed with an appropriately engineered vector that includesexpression control elements (e.g., promoter, enhancer, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker gene.Following the introduction of the foreign DNA, cells are allowed to growfor 1-2 days in an enriched media, and are then switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells that have stably integratedthe plasmid into their chromosomes to grow and form foci which in turncan be cloned and expanded into cell lines. Methods for producing stablecell lines with a high yield are known in the art and reagents aregenerally available commercially. Transient expression can also becarried out by using methods known in the art. Transient transfection isa process in which the nucleic acid introduced into a cell does notintegrate into the genome or chromosomal DNA of that cell and ismaintained as an extra-chromosomal element in the cell (e.g., as anepisome).

A cell line, either stable or transiently transfected, is maintained incell culture medium and conditions known in the art resulting in theexpression and production of the binding molecule. Cell culture mediacan be based on commercially available media formulations, including,for example, DMEM or Ham's F12. In addition, the cell culture media canbe modified to support increases in both cell growth and biologicprotein expression. As used herein, the terms “cell culture medium,”“culture medium,” and “medium formulation” refer to a nutritive solutionfor the maintenance, growth, propagation, or expansion of cells in anartificial in vitro environment outside of a multicellular organism ortissue. Cell culture medium may be optimized for a specific cell cultureuse, including cell culture growth medium which is formulated to promotecellular growth or cell culture production medium which is formulated topromote recombinant protein production. The terms nutrient, ingredient,and component are used interchangeably herein to refer to theconstituents that make up a cell culture medium. Cell lines can bemaintained using a fed batch method. As used herein, “fed batch method,”refers to a method by which a cell culture is supplied with additionalnutrients after first being incubated with a basal medium. For example,a fed batch method may include adding supplemental media according to adetermined feeding schedule within a given time period. Thus, a “fedbatch cell culture” refers to a cell culture wherein the cells,typically mammalian, and culture medium are supplied to the culturingvessel initially and additional culture nutrients are fed, continuouslyor in discrete increments, to the culture during culturing, with orwithout periodic cell and/or product harvest before termination ofculture.

Cell culture media and the nutrients contained therein are known to oneof skilled in the art. The cell culture medium may include a basalmedium and at least one hydrolysate, e.g., soy-based hydrolysate, ayeast-based hydrolysate, or a combination of the two types ofhydrolysates resulting in a modified basal medium. The additionalnutrients may include only a basal medium, such as a concentrated basalmedium, or may include only hydrolysates, or concentrated hydrolysates.Suitable basal media include, but are not limited to Dulbecco's ModifiedEagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), BasalMedium Eagle (BME), RPMI 1640, F-10, F-12, α-Minimal Essential Medium(α-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g.,CHO protein free medium (Sigma) or EX-CELL™ 325 PF CHO Serum-Free Mediumfor CHO Cells Protein-Free (SAFC Bioscience), and Iscove's ModifiedDulbecco's Medium. Other examples of basal media which may be used inthe invention include BME Basal Medium (Gibco-Invitrogen; see alsoEagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's ModifiedEagle Medium (DMEM, powder) (Gibco-Invitrogen (#31600); see alsoDulbecco and Freeman (1959) Virology. 8:396; Smith et al. (1960)Virology. 12:185. Tissue Culture Standards Committee, In Vitro 6:2, 93);CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker et al.(1957) Special Publications, N.Y. Academy of Sciences, 5:303).

The basal medium may be serum-free, meaning that the medium contains noserum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or anyother animal-derived serum known to one skilled in the art) or animalprotein free media or chemically defined media.

The basal medium may be modified in order to remove certainnon-nutritional components found in standard basal medium, such asvarious inorganic and organic buffers, surfactant(s), and sodiumchloride. Removing such components from basal cell medium allows anincreased concentration of the remaining nutritional components, and mayimprove overall cell growth and protein expression. In addition, omittedcomponents may be added back into the cell culture medium containing themodified basal cell medium according to the requirements of the cellculture conditions. The cell culture medium may contain a modified basalcell medium, and at least one of the following nutrients, an ironsource, a recombinant growth factor; a buffer; a surfactant; anosmolarity regulator; an energy source; and non-animal hydrolysates. Inaddition, the modified basal cell medium may optionally contain aminoacids, vitamins, or a combination of both amino acids and vitamins. Amodified basal medium may further contain glutamine, e.g, L-glutamine,and/or methotrexate.

Purification and Isolation

Once a binding molecule has been produced, it may be purified by methodsknown in the art for purification of an immunoglobulin molecule, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigens Protein A or Protein G, and sizingcolumn chromatography), centrifugation, differential solubility, or byany other standard technique for the purification of proteins. Further,the binding molecules of the invention may be fused to heterologouspolypeptide sequences (referred to herein as “tags”) to facilitatepurification.

Uses

Binding molecules of the invention can be used in a number of ways. Forexample, antibodies of the invention can be used to bind to VEGF-A andthereby reduce at least one biological activity of VEGF-A. Moreparticularly, the antibodies of the invention can be used to bind toVEGF-165 and thereby reduce at least one biological activity ofVEGF-165, which may include a reduction in activation or phosphorylationof its receptor, a reduction in angiogenesis in connection with cellulardysregulation, a reduction in tumor growth, a reduction in tumor volume,and/or reduction in tumor growth and tumor volume.

Exemplary Embodiments

An embodiment of the invention relates to a binding molecule comprisingheavy chain complementarity determining regions 1-3 (i.e., HCDR1, HCDR2,and HCDR3) and light chain complementarity determining regions 1-3(i.e., LCDR1, LCDR2, and LCDR3) of an antibody described herein.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF121.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF189.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF121 and VEGF189.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces human VEGFR2phosphorylation, murine VEGFR2 phosphorylation, or both human and murineVEGFR2 phosphorylation.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces angiogenesis.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces tumor growth, reduces tumorvolume, or reduces tumor growth and tumor volume as a result of beingprovided to a subject having a tumor.

Another embodiment relates to a binding molecule comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule has one or more or any combinationof the characteristics described herein, including binding VEGF165,binding VEGF165 with greater affinity compared to VEGF121, bindingVEGF165 with greater affinity compared to VEGF189, binding VEGF165 withgreater affinity compared to VEGF121 and VEGF189, reducing human VEGFR2phosphorylation, murine VEGFR2 phosphorylation, or both human and murineVEGFR2 phosphorylation, reducing angiogenesis, or reducing tumor growth,reducing tumor volume, or reducing tumor growth and tumor volume as aresult of being provided to a subject having a tumor.

An embodiment of the invention relates to a binding molecule comprisinga heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 and alight chain variable domain comprising LCDR1, LCDR2, and LCDR3 of anantibody described herein.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and a lightchain variable domain comprising LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein, wherein the binding molecule binds VEGF165.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and a lightchain variable domain comprising LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein, wherein the binding molecule binds VEGF165 withgreater affinity compared to VEGF121.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and a lightchain variable domain comprising LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein, wherein the binding molecule binds VEGF165 withgreater affinity compared to VEGF189.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and a lightchain variable domain comprising LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein, wherein the binding molecule binds VEGF165 withgreater affinity compared to VEGF121 and VEGF189.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and a lightchain variable domain comprising LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein, wherein the binding molecule reduces human VEGFR2phosphorylation, murine VEGFR2 phosphorylation, or both human and murineVEGFR2 phosphorylation.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and comprisinga light chain variable domain comprising LCDR1, LCDR2, and LCDR3 of anantibody described herein, wherein the binding molecule reducesangiogenesis.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and comprisinga light chain variable domain comprising LCDR1, LCDR2, and LCDR3 of anantibody described herein, wherein the binding molecule reduces tumorgrowth, reduces tumor volume, or reduces tumor growth and tumor volumeas a result of being provided to a subject having a tumor.

Another embodiment relates to a binding molecule comprising a heavychain variable domain comprising HCDR1, HCDR2, and HCDR3 and comprisinga light chain variable domain comprising LCDR1, LCDR2, and LCDR3 of anantibody described herein, wherein the binding molecule has one or moreor any combination of the characteristics described herein, includingbinding VEGF165, binding VEGF165 with greater affinity compared toVEGF121, binding VEGF165 with greater affinity compared to VEGF189,binding VEGF165 with greater affinity compared to VEGF121 and VEGF189,reducing human VEGFR2 phosphorylation, murine VEGFR2 phosphorylation, orboth human and murine VEGFR2 phosphorylation, reducing angiogenesis, orreducing tumor growth, reducing tumor volume, or reducing tumor growthand tumor volume as a result of being provided to a subject having atumor.

An embodiment of the invention relates to a binding molecule comprisinga full-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF121.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF189.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF121 andVEGF189.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces human VEGFR2 phosphorylation, murine VEGFR2phosphorylation, or both human and murine VEGFR2 phosphorylation.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces angiogenesis.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces tumor growth, reduces tumor volume, or reduces tumorgrowth and tumor volume as a result of being provided to a subjecthaving a tumor.

Another embodiment relates to a binding molecule comprising afull-length antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule has one or more or any combination of the characteristicsdescribed herein, including binding VEGF165, binding VEGF165 withgreater affinity compared to VEGF121, binding VEGF165 with greateraffinity compared to VEGF189, binding VEGF165 with greater affinitycompared to VEGF121 and VEGF189, reducing human VEGFR2 phosphorylation,murine VEGFR2 phosphorylation, or both human and murine VEGFR2phosphorylation, reducing angiogenesis, or reducing tumor growth,reducing tumor volume, or reducing tumor growth and tumor volume as aresult of being provided to a subject having a tumor.

An embodiment of the invention relates to a binding molecule comprisinga full-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 andLCDR1, LCDR2, and LCDR3 of an antibody described herein.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF121.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF189.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule binds VEGF165 with greater affinity compared to VEGF121 andVEGF189.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces human VEGFR2 phosphorylation, murine VEGFR2phosphorylation, or both human and murine VEGFR2 phosphorylation.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces angiogenesis.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule reduces tumor growth, reduces tumor volume, or reduces tumorgrowth and tumor volume as a result of being provided to a subjecthaving a tumor.

Another embodiment relates to a binding molecule comprising afull-length IgG1 antibody comprising HCDR1, HCDR2, and HCDR3 and LCDR1,LCDR2, and LCDR3 of an antibody described herein, wherein the bindingmolecule has one or more or any combination of the characteristicsdescribed herein, including binding VEGF165, binding VEGF165 withgreater affinity compared to VEGF121, binding VEGF165 with greateraffinity compared to VEGF189, binding VEGF165 with greater affinitycompared to VEGF121 and VEGF189, reducing human VEGFR2 phosphorylation,murine VEGFR2 phosphorylation, or both human and murine VEGFR2phosphorylation, reducing angiogenesis, or reducing tumor growth,reducing tumor volume, or reducing tumor growth and tumor volume as aresult of being provided to a subject having a tumor.

An embodiment of the invention relates to a binding molecule which is afull-length antibody, including a full-length IgG1 antibody, comprisingHCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibodydescribed herein.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF121.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF189.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule binds VEGF165 with greater affinitycompared to VEGF121 and VEGF189.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces human VEGFR2phosphorylation, murine VEGFR2 phosphorylation, or both human and murineVEGFR2 phosphorylation.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces angiogenesis.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule reduces tumor growth, reduces tumorvolume, or reduces tumor growth and tumor volume as a result of beingprovided to a subject having a tumor.

Another embodiment relates to a binding molecule which is a full-lengthantibody, including a full-length IgG1 antibody, comprising HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of an antibody describedherein, wherein the binding molecule has one or more or any combinationof the characteristics described herein, including binding VEGF165,binding VEGF165 with greater affinity compared to VEGF121, bindingVEGF165 with greater affinity compared to VEGF189, binding VEGF165 withgreater affinity compared to VEGF121 and VEGF189, reducing human VEGFR2phosphorylation, murine VEGFR2 phosphorylation, or both human and murineVEGFR2 phosphorylation, reducing angiogenesis, or reducing tumor growth,reducing tumor volume, or reducing tumor growth and tumor volume as aresult of being provided to a subject having a tumor.

In a specific embodiment, there is an antibody comprising an HCDR1,HCDR2, and HCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2,and HCDR3 and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84,respectively.

In another specific embodiment, there is an antibody comprising a heavychain and a light chain comprising SEQ ID NOs: 73 and 77, respectively.

In another specific embodiment, there is an antibody comprising a heavychain amino acid sequence comprising SEQ ID NO: 71 and a light chainamino acid sequence comprising SEQ ID NO: 75.

In another specific embodiment, there is an antibody comprising anHCDR1, HCDR2, and HCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1,HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84,respectively, and wherein the antibody is a monoclonal antibody.

In another specific embodiment, there is a nucleic acid sequencecomprising polynucleotides encoding an antibody comprising an HCDR1,HCDR2, and HCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2,and HCDR3 and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84,respectively.

In another specific embodiment, there is a vector comprisingpolynucleotides encoding an antibody comprising an HCDR1, HCDR2, andHCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84, respectively.

In another specific embodiment, there is a cell comprising a vectorcomprising polynucleotides encoding an antibody comprising an HCDR1,HCDR2, and HCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2,and HCDR3 and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84,respectively.

In another specific embodiment, there is a method of making an antibodycomprising culturing a cell comprising a vector comprisingpolynucleotides encoding an antibody comprising an HCDR1, HCDR2, andHCDR3 and an LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3and LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 79-84, respectively.

In another specific embodiment, there is a method of reducingangiogenesis comprising providing an antibody to a subject wherein theantibody comprises an HCDR1, HCDR2, and HCDR3 and an LCDR1, LCDR2, andLCDR3, wherein HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3comprise SEQ ID NOs: 79-84, respectively.

Sequences SEQ ID NO SEQUENCE DESCRIPTION 1EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYEMYWVRQAAmino acid sequence of the heavyPGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTLYL chain of E06QMNSLRAEDTAVYYCATPLYSSDGLSAGDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 2GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGG Nucleotide sequence of the heavyTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCA chain of E06GCGGCTTCACCTTCAGCTGGTACGAGATGTACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCAGCATCAGCCCCAGCGGCGGCTGGACCATGTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCACCCCCCTGTACAGCAGCGACGGCCTGAGCGCCGGCGACATCTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGGGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACAGGGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGCCCTGCCCGCCCCCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCAGGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCCCGGCAAG 3EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYEMYWVRQAAmino acid sequence of the heavyPGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTLYL chain variable domain of E06QMNSLRAEDTAVYYCATPLYSSDGLSAGDIWGQGTMVTVS S 4GAGGTGCAGCTGCTGGAGAGCGGCGGCGGCCTGGTGCAG Nucleotide sequence of the heavyCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGC chain variable domain of E06TTCACCTTCAGCTGGTACGAGATGTACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCAGCATCAGCCCCAGCGGCGGCTGGACCATGTACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCACCCCCCTGTACAGCAGCGACGGCCTGAGCGCCGGCGACATCTGGGGCCAGGGCACCATG GTGACCGTGAGCAGC 5DIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPAmino acid sequence of the lightGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF chain of E06ATYYCQQSYSTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 6GACATCCAGATGACCCAGAGCCCCGCCACCCTGA Nucleotide sequence of the lightGCCTGAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGG chain of E06CCAGCCAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCA CCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGG GGCGAGTGC 7DIQMTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPAmino acid sequence of the lightGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF chain variable domain of E06ATYYCQQSYSTPSFGQGTRLEIT 8 GACATCCAGATGACCCAGAGCCCCGCCACCCTGAGCCTGNucleotide sequence of the light AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCchain variable domain of E06 CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 9 WYEMYAmino acid sequence of HCDR1 of E06 10 SISPSGGWTMYADSVKGAmino acid sequence of HCDR2 of E06 11 PLYSSDGLSAGDIAmino acid sequence of HCDR3 of E06 12 RASQSVSSSYLAAmino acid sequence of LCDR1 of E06 13 GASSRATAmino acid sequence of LCDR2 of E06 14 QQSYSTPSAmino acid sequence of LCDR3 of E06 15 SAME AS E06Amino acid sequence of the heavy chain of E06 germline M4 16 SAME AS E06Nucleotide sequence of the heavy chain of E06 germline M4 17 SAME AS E06Amino acid sequence of the heavy chain variable domain of E06germline M4 18 SAME AS E06 Nucleotide sequence of the heavychain variable domain of E06 germline M4 19EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of E06 germline M4VYYCQQSYSTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 20GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of E06 germline M4CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 21EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain variable domain of E06VYYCQQSYSTPSFGQGTRLEIT germline M4 22GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain variable domain of E06CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAG germline M4AAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 23 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of E06 germline M4 24 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of E06 germline M4 25 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of E06 germline M4 26 SAME AS E06 LCDR1Amino acid sequence of LCDR1 of E06 germline M4 27 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of E06 germline M4 28 SAME AS E06 LCDR3Amino acid sequence of LCDR3 or E06 germline M4 29 SAME AS E06Amino acid sequence of the heavy chain of D04 30 SAME AS E06Nucleotide sequence of the heavy chain of D04 31 SAME AS E06Amino acid sequence of the heavy chain variable domain of D04 32SAME AS E06 Nucleotide sequence of the heavychain variable domain of D04 33EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of D04VYYCQQSYSTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 34GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of D04CAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 35EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain variable domain of D04VYYCQQSYSTPSFGQGTRLEIT 36 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGNucleotide sequence of the light AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCchain variable domain of D04 CAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 37 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of D04 38 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of D04 39 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of D04 40 RASQSVHSSYLAAmino acid sequence of LCDR1 of D04 41 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of D04 42 SAME AS E06 LCDR3Amino acid sequence of LCDR3 of D04 43 SAME AS E06Amino acid sequence of the heavy chain of J05 44 SAME AS E06Nucleotide sequence of the heavy chain of J05 45 SAME AS E06Amino acid sequence of the heavy chain variable domain of J05 46SAME AS E06 Nucleotide sequence of the heavychain variable domain of J05 47EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of J05VYYCQQSYRTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 48GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of J05CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 49EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain variable domain of J05VYYCQQSYRTPSFGQGTRLEIT 50 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGNucleotide sequence of the light AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCchain variable domain of J05 CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 51 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of J05 52 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of J05 53 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of J05 54 SAME AS E06 LCDR1Amino acid sequence of LCDR1 of J05 55 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of J05 56 QQSYRTPSAmino acid sequence of LCDR3 of J05 57 SAME AS E06Amino acid sequence of the heavy chain of I20 58 SAME AS E06Nucleotide sequence of the heavy chain of I20 59 SAME AS E06Amino acid sequence of the heavy chain variable domain of I20 60SAME AS E06 Nucleotide sequence of the heavychain variable domain of I20 61EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of I20VYYCQQDYSTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 62GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of I20CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 63EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain variable domain of I20VYYCQQDYSTPSFGQGTRLEIT 64 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGNucleotide sequence of the light AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCchain variable domain of I20 CAGAGCGTGAGCAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACTACAGCACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 65 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of I20 66 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of I20 67 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of I20 68 SAME AS E06 LCDR1Amino acid sequence of LCDR1 of I20 69 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of I20 70 QQDYSTPSAmino acid sequence of LCDR3 of I20 71 SAME AS E06Amino acid sequence of the heavy chain of H1R 72 SAME AS E06Nucleotide sequence of the heavy chain of H1R 73 SAME AS E06Amino acid sequence of the heavy chain variable domain of H1R 74SAME AS E06 Nucleotide sequence of the heavychain variable domain of H1R 75EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of H1RVYYCQQSYRTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 76GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of H1RCAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 77EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain variable domain of H1RVYYCQQSYRTPSFGQGTRLEIT 78 GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGNucleotide sequence of the light AGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGCchain variable domain of H1R CAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGCTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 79 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of H1R 80 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of H1R 81 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of H1R 82 RASQSVHSSYLAAmino acid sequence of LCDR1 of H1R 83 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of H1R 84 QQSYRTPSAmino acid sequence of LCDR3 of H1R 85EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYEMYWVRQAAmino acid sequence of the heavyPGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTLYL chain of H1RKQMNSLRAEDTAVYYCATPLYSSDGLSAGDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK 86GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCNucleotide sequence of the heavyCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTC chain of H1RKACTTTCTCTTGGTACGAGATGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCTGGTGGCTGGACTATGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGACCCCCTTGTATAGCAGTGACGGGCTTTCGGCGGGGGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCGTCGACCAAGGGCCCATCCGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCCTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCTCCGGGTAAA 87EVQLLESGGGLVQPGGSLRLSCAASGFTFSWYEMYWVRQAAmino acid sequence of the heavyPGKGLEWVSSISPSGGWTMYADSVKGRFTISRDNSKNTLYL chain variable domain of H1RKQMNSLRAEDTAVYYCATPLYSSDGLSAGDIWGQGTMVTVS S 88GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCNucleotide sequence of the heavyCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTC chain variable domain of H1RKACTTTCTCTTGGTACGAGATGTATTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTCTCCTTCTGGTGGCTGGACTATGTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGACCCCCTTGTATAGCAGTGACGGGCTTTCGGCGGGGGATATCTGGGGCCAAGGGACAATGGTCACCGTC TCAAGC 89EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of H1RKVYYCQQSYRTPSFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 90GAGATCGTGCTGACCCAGTCTCCAGCCACCCTCTCTTTGTNucleotide sequence of the light CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCchain of H1RK AGAGTGTTCACAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTACTACTGTCAACAGAGTTACCGCACCCCTTCCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAA CAGGGGAGAGTGT 91EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAchain variable domain of H1RK VYYCQQSYRTPSFGQGTRLEIK 92GAGATCGTGCTGACCCAGTCTCCAGCCACCCTCTCTTTGTNucleotide sequence of the light CTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCchain variable domain of H1RK AGAGTGTTCACAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTACTACTGTCAACAGAGTTACCGCACCCCTTCCTTCGGCCAAGGGACACGACTGGAGA TTAAA 93 WYEMYAmino acid sequence of HCDR1 of H1RK 94 SISPSGGWTMYADSVKGAmino acid sequence of HCDR2 of H1RK 95 PLYSSDGLSAGDIAmino acid sequence of HCDR3 of H1RK 96 RASQSVHSSYLAAmino acid sequence of LCDR1 of H1RK 97 GASSRATAmino acid sequence of LCDR2 of H1RK 98 QQSYRTPSAmino acid sequence of LCDR3 of H1RK 99 SAME AS E06Amino acid sequence of the heavy chain of H1DR 100 SAME AS E06Nucleotide sequence of the heavy chain of H1DR 101 SAME AS E06Amino acid sequence of the heavy chain variable domain of H1DR 102SAME AS E06 Nucleotide sequence of the heavychain variable domain of H1DR 103EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFA chain of H1DRVYYCQQDYRTPSFGQGTRLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 104GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain of H1DRCAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTGGAGATCACCAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAG CTTCAACAGGGGCGAGTGC 105EIVLTQSPATLSLSPGERATLSCRASQSVHSSYLAWYQQKPGAmino acid sequence of the lightQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAchain variable domain of H1DR VYYCQQDYRTPSFGQGTRLEIT 106GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTG Nucleotide sequence of the lightAGCCCCGGCGAGAGGGCCACCCTGAGCTGCAGGGCCAGC chain variable domain of H1DRCAGAGCGTGCACAGCAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGGCTGCTGATCTACGGCGCCAGCAGCAGGGCCACCGGCATCCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGGCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCAGCAGGACTACAGGACCCCCAGCTTCGGCCAGGGCACCAGGCTG GAGATCACC 107 SAME AS E06 HCDR1Amino acid sequence of HCDR1 of H1DR 108 SAME AS E06 HCDR2Amino acid sequence of HCDR2 of H1DR 109 SAME AS E06 HCDR3Amino acid sequence of HCDR3 of H1DR 110 RASQSVHSSYLAAmino acid sequence of LCDR1 of H1DR 111 SAME AS E06 LCDR2Amino acid sequence of LCDR2 of H1DR 112 QQDYRTPSAmino acid sequence of LCDR3 of H1DR

EXAMPLES

For the experiments described herein various antibodies were usedincluding, Avastin® (Ferrara, N et al. Biochem Biophys Res Comm,333:328-335, 2005), G6-31 (Liang, W C et al. J Biol Chem, 281: 951-961,2006), B20-4.1 (Liang, W C et al. J Biol Chem, 281: 951-961, 2006), andan isotype control, designated R347, as a monospecific or a bispecificantibody as needed. An anti-VEGF IgG1 antibody capable of binding allVEGF isoforms that is not cross-reactive with mouse can used as apositive control for some binding and functional studies. Where crossreactivity to mouse VEGF is needed the antibodies G6-31 and B20-4.1 canbe used as a positive control.

Example 1: Identification of Anti-VEGF Antibodies from Phage AntibodyDisplay Libraries

Solution phage panning was applied to isolate anti-VEGF antibodies fromantibody phage libraries. The phage libraries were incubated withbiotinylated VEGF165 (PeproTech, N.J.) at the final concentration of 4μg/ml (human VEGF165 for first and third round and mVEGF164 for thesecond and fourth round panning). After incubation for 30 minutes, theVEGF bound phages were captured from the solution by adding straptavidinbeads (Invitrogen), which were prewashed with phosphate buffer saline(PBS) and blocked with 3% bovine serum albumin (BSA) in PBS. The beadscaptured phages were eluted with 100 mM TEA buffer, neutralized with 1 MTris-HCI (pH8.0), and then 1 ml of eluted phage were used to infect 5 mlof log phase TG1 and 4 ml of 2YT medium (Teknova) for phageamplification. After incubation at 37° C. for 30 minutes in water bath,the cells were spun down at 4000 g, resuspended in 2YT, spread on 2YTagar plates (Teknova) with 100 μg/ml of carbenicillin and 2% glucose,and incubated at 30° C. overnight. On the second day, the colonies werecollected from the agar plates, inoculated into 2YT medium at the finaldensity of OD600=0.1, grown at 37° C. until log phase, infected withhelper phage (Invitrogen) and then let it grow overnight in 2YT withcarbenicillin (Invitrogen) and kanamycin (Sigma) at 30° C. to generatehigh titer phages. The amplified phages were precipitated with PEG8000,resuspended in PBS and used for the next round of panning using thestandard procedure. A total of four rounds of panning were applied toisolate VEGF specific antibodies. A significant enrichment of outputphage numbers was observed from 2×10⁵ pfu in the first round to 4×10⁷pfu in the fourth round of panning.

Example 2: Screening of VEGF Specific Antibodies

Specificity of individual phage Fab from the fourth round of panning wasassessed by phage ELISA. The 96-well microtiter plates were coated withhuman VEGF165 at a concentration of 5 μg/ml in PBS at 40° C. overnight.After being washed three times with PBS, the wells were blocked with theblocking buffer (4% skimmed milk in PBS) for 1 h at 37° C. Then, 50μl/well phage was added and incubated for 1 h at 37° C. After washing,50 μl of horseradish peroxidase (HRP)-conjugated mouse anti-M13(Amersham Pharmacia) in blocking buffer with 1:1,000 dilution was addedfor 30 minutes at 37° C. For detection, 50 μl/well of SureBlue ReserveTMB substrate (KPL) for 5 minutes at room temperature and the reactionwas stopped by adding 50 μL of TMB Stop Solution (KPL). The absorbancewas read at 450 nm. After four rounds of panning, more than 50% ofclones are VEGF specific with absorbance higher than 1, which is 20-foldhigher than the nonspecific background reading approximately 0.05.Representative data is show in Table 1.

TABLE 1 1 2 3 4 5 6 7 8 9 10 11 12 0.091 2.276 2.406 0.111 0.116 0.1160.087 2.534 0.059 2.478 0.098 0.193 0.091 2.755 3.161 1.508 0.054 3.1660.062 0.066 0.061 3.2 2.354 1.971 2.328 0.078 2.697 2.919 3.216 3.2583.133 2.948 2.859 2.6 1.314 2.595 2.209 3.056 0.084 3.139 3.181 0.0553.336 2.472 3.074 0.062 2.741 0.073 0.05 0.052 0.053 3.02 3.57 0.0490.047 2.862 2.761 2.609 0.051 0.05 0.05 0.05 0.053 2.861 3.469 3.1550.057 0.387 1.325 0.052 3.067 2.446 1.963 2.641 3.27 3.349 3.246 0.0473.509 2.727 0.046 2.67 2.679 0.053 0.044 0.05 3.105 0.053 3.215 2.4830.059 3.004 2.294 2.994 3.091 1.336

Example 3-Binding of Clone E06 to Human and Mouse VEGF

After initial phage screening, one clone (E06) with cross-reactivity tomouse VEGF164 was further converted and expressed as the full length IgGusing standard molecular biology materials and methods. The 96-wellmicrotiter plates (Corning) were coated with 50 μl/well of human andmouse VEGF165 or VEGF121 at a concentration of 5 μg/ml in PBS at 4° C.overnight. After being washed three times with PBS containing 0.1%Tween-20 (PBST), the wells were blocked with blocking buffer (4% skimmedmilk in PBS). After 1 h incubation at room temperature, the plates werewashed with PBST and a 2-fold serial dilution of the antibodies(starting from 8 nM) in blocking buffer were added and incubated for 1hour at room temperature. The antibody solution was removed by washingwith PBST followed by 1 hour incubation at room temperature with a1:3000 dilution of an anti-human-Fc-HRP antibody (Thermo Scientific)prepared in PBST. Binding was visualized with the addition of 50 μL ofSureBlue Reserve TMB substrate (KPL) for 5 minutes at room temperatureand the reaction was stopped by adding 50 μL of TMB Stop Solution (KPL).The absorbance at 450 nm was measured using a microtiter plate reader.The data were analyzed using Prism 5 software (GraphPad).

Similar binding activity of antibody E06 to human VEGF165 and mouseVEGF164 was observed with an EC50 of 0.048 nM and 0.049 nM,respectively. As expected, Avastin® (an anti-VEGF antibody) showedstrong binding to human VEGF165 but no detectable binding to mouseVEGF164. The isotype control antibody R347 did not bind either human ormouse forms of VEGF. Representative data are shown in FIG. 1 .

To evaluate whether antibody E06 binds to a different epitope thanAvastin® (an anti-VEGF antibody), an ELISA assay was conducted usinghuman VEGF121. Avastin® (an anti-VEGF antibody) showed very strongbinding to human VEGF121. However, no binding was deteceted for E06,indicating antibody E06 binds to a different epitope than Avastin® (ananti-VEGF antibody). The isotype control antibody R347 also did notdemonstrate any binding to human VEGF121. Representative data are shownin FIG. 2 .

Example 4-Germline of Clone E06

Framework sequences of clone E06 were engineered to match to its closestgermline sequences. A sequence analysis against the IgG germline genedatabase showed that the E06 VL sequence best matches to the germlinegene VK3-NL5*01 with 4 amino acid differences at positions 1, 3, 4 and86 of the VL. Although the VH sequence is identical to the germline geneVH3-23*2, the T98 differs from most conserved germline amino acid of Ror K at this position. To germline E06, four variants were designed andexpressed. These variants substitute partially or totally with thegermline gene encoded amino acids at the positions that E06 sequencediffers from the best matched germline genes. For instance, M1 containsD1E/Q3V/M4L substitutions in the VL and T98R substitution in the VH; M4contains D1E/Q3V/M4L/T86V substitutions in the VL; M7 containsD1E/Q3V/M4L/T86V substitutions in the VL and T98R substitution in theVH; and F1 contains the D1E/Q3V/M4L substitutions in the VL, where thefirst letter represents the one letter amino acid code of the originaland the second letter represents the one letter amino acid code of thegermline sequence. FIG. 3 shows the sequence alignment of the parentalE06 and the most homologous germline genes.

The germline variants were expressed and purified as Fabs and theirbinding to the recombinant VEGF165 was determined by ELISA. The bindingresults showed that the germline variants M4 and F1, which contain thefull or partial VL germline amino acid substitutions retained E06 WT Fabbinding activity. In addition, M4 showed similar activity compared tothe WT E06 Fab in pVEGFR2 assay using HUVECs. On the other hand,germline variants M1 and M7 containing the VH germline amino acidsubstitution at position T98 demonstrated drastically reduced bindingand pVEGFR2 phosphorylation, indicating that T98 participated in bindingand activity and must be retained. The germline clone, M4, was used asthe template for further affinity optimization.

Example 5-Affinity Optimization

Each amino acid of all 6 CDRs of germline clone M4 was individuallymutated to the other 20 amino acids using a hybridization mutagenesismethod (Kunkel, Proc. Natl. Acad. Sci. USA Vol. 82, pp. 488-492, 1985).Two sets of DNA primers, one containing a NSS codon encoding 8 aminoacids and the other containing a NWS codon encoding 12 different aminoacids, were used to introduce mutations to each targeted CDR position.The individual degenerate primers were used in hybridization mutagenesisreactions. Briefly, each degenerate primer was phosphorylated and usedin a 10:1 ratio with the uridinylated M4 Fab ssDNA. The mixture washeated to 95° C. then cooled down to 55° C. over 1 hour. Thereafter, T4ligase and T7 DNA polymerase were added and the mix was incubated for1.5 hours at 37° C. Synthesis products for VH and VL CDRs were pooledrespectively; however, NSS and NWS libraries were kept separate andscreened independently. Typically, 1 μL of the pooled library DNA waselectroporated into XL1-Blue for plaque formation on XL1-Blue bacteriallawn or for production of Fab fragments (Wu H, An LL. Tailoring kineticsof antibodies using focused combinatorial libraries. Methods Mol Biol2003; 207:213-33). These mutants were then screened for activity.

The primary screen consisted of a single point ELISA (SPE) assay whichwas carried out using culture supernatant of bacteria grown in 96-wellplates (deep well) and infected with individual recombinant M13 clonesas described elsewhere (Wu H, An LL. Tailoring kinetics of antibodiesusing focused combinatorial libraries. Methods Mol Biol 2003;207:213-33). Briefly, this capture ELISA involved coating individualwells of a 96-well Maxisorp Immunoplate with approximately 50 ng of asheep anti-human Fd antibody (Biodesign International, ME) in acarbonate buffer at pH 8.5 overnight at 4° C. The next day, the platewas blocked with 3% BSA in PBS buffer for 1 hour at room temperature.Fab supernatant was then added to the plate and incubated at roomtemperature for 1 hour. After washing, the biotinylated VEGF165 proteinwas added to the well and the mixture was incubated for 1.5 hours atroom temperature. This was followed by incubation withneutravidin-horseradish peroxydase (HRP) conjugate (Pierce, Ill.) forapproximately 40 minutes at room temperature. HRP activity was detectedwith tetra-methyl-benzidine (TMB) substrate and the reaction quenchedwith 0.2 M H2SO4. Plates were read at 450 nm.

Clones exhibiting an optical density (OD) signal at 450 nm greater thanthe parental clone M4 Fab were picked and regrown (15 mL) (Wu H, An LL.Tailoring kinetics of antibodies using focused combinatorial libraries.Methods Mol Biol 2003; 207:213-33) and re-assayed by ELISA (as describedabove) in duplicate to confirm positive results. Clones that repeatedlyexhibited a signal greater than that of the M4 Fab were sequenced. TheFab protein concentration of each clone that had a CDR change was thendetermined by a quantitative Fab ELISA, where a Fab with knownconcentration was used as a reference. The Fab concentration wasdetermined by comparing the ELISA signals with the signals generated bythe reference Fab. The binding assay was repeated once more for allpositive variants under normalized Fab concentrations in order todetermine the relative binding affinity of the mutant Fabs and theparental M4 Fab.

Many point mutations showed binding improvements over M4 to VEGF165.Among those mutants, D04, J05, and I20 showed in excess of 10-foldimprovement in EC50 compared to germline E06 as Fabs. Representativedata are shown in FIG. 4 . Sequence analysis revealed that amino acidsubstitutions that benefit to the VEGF165 binding are found in the VL,especially in the VL-CDR3. For example, a point mutation S94R in mutantJ05 improved binding approximately 25-fold over E06, indicating keycontributions of this amino acid in binding.

The point mutants demonstrating improved binding were then combinedusing site-directed mutagenesis methods. The combination mutants wereexpressed as Fabs and IgGs and tested in a VEGF165 ELISA. Representativedata are shown in FIG. 5 .

Combination mutants showed significantly higher binding than a pointmutant, J05, in an ELISA assay. The apparent binding affinity of thecombination mutants was improved approximately 3- to 10-fold over J05 asFabs. Similarly, all combination mutants tested as IgGs showedsignificant binding improvement compared to the parental clone E06. Theequilibrium binding constants (KD) of the affinity optimized clones weremeasurements using KinExA. Representative data are shown in Table 2.Sequences of point mutants as well as combination mutants are shown inTable 3.

TABLE 2 K_(D), pM (95% CI) FoldΔ K_(D) IgG (Std. Aff. model - ref[VEGF165]) vs. EO6-wt H1R 23.2 (10.8-40.9) 109  J05 220.3 (179.1-268.4)11 Wt (E06) 2520 (1800-4260) — Avastin ® 99.9 (71.1-137.0) 25

TABLE 3 (The CDR 1 column discloses SEQ ID NOS113-114, 113, 113-114, and 114, respectively, inorder of appearance, and the CDR 3 columndiscloses 115, 115-117, 116, and 118,respectively, in order of appearance) VL CDR1 CDR3 E06R A S Q S V S S S Y L A Q Q S Y S T P S D04 R A S Q S V  H  S S Y L AQ Q S Y S T P S J05 R A S Q S V S S S Y L A Q Q S Y  R  T P S I20R A S Q S V S S S Y L A Q Q  D  Y S T P S H1R R A S Q S V  H  S S Y L AQ Q S Y  R  T P S H1DR R A S Q S V  H  S S Y L A Q Q  D  Y  R  T P S

Example 6-Measurement of Kd for the Binding of Avastin, E06 and AffinityOptimized E06 Variants to Human VEGF165

Equilibrium binding constant (KD) measurements were performed on KinExA3000 and 3200 instruments (Sapidyne Instruments, Boise, Id.). HumanVEGF165 (huVEGF) protein was coated onto UltraLink® Biosupport beads(PIERCE, Rockford, Ill.) at concentrations of 2 ug/mL, 3 ug/mL, and 30ug/mL in coating buffer (50 mM sodium carbonate buffer, pH 9). Coatedbeads were then separated (spin) from unreacted huVEGF protein solution,and blocked with 1M Tris, pH 8, containing BSA at 10 mg/mL), forapproximately 15 minutes at room temperature. After this, the beadslurry was spun to remove the blocking solution, and then the block stepwas repeated for approximately 2 hours using fresh block buffer, andstored at 4° C. until used. Prior to use, the huVEGF-coated beads weretransferred to a bead vial, resuspended in approximately 27 mLs ofinstrument buffer (10 mM HEPES+300 mM NaCl+5 mM CaCl₂+0.05% P20+0.02%NaN₃, pH8), and affixed to the KinExA instrument. For the KDmeasurements, separate solutions of antibodies were prepared at 100 pMand 2.5 nM concentrations in instrument buffer containing BSA (mg/mL),then dispensed into two separate series of 13 tubes. Theseconcentrations of mAbs were chosen to allow each KD measurement to bemade under both receptor- and KD-controlled conditions, leading to morerigorous estimations of reagent activity and affinity, respectively.Owing to its relatively weak KD, a 3rd concentration series (50 nM) formAb E06-wt was also prepared to satisfy the requirement for a fullyreceptor-controlled measurement. Two-fold serial dilutions of huVEGFprotein were then titrated across 9 of the tubes in each mAb series,followed by two additional 10-fold-dilutions, leaving one tube as themAb-only, “zero” control. In so doing, this yielded concentrationseries' of huVEGF protein that ranged from 488 fM-25 nM (100 pM mAbexperiments), 3.91 pM-200 nM (2.5 nM mAb experiments), and 9.77 pM-500nM (50 nM mAb experiment). Based on theory curve simulations availablethrough the vendor software (Sapidyne Instruments, Boise, Idaho), themixtures were incubated 1-4 days at room temperature to allow binding toreach equilibrium. At the end of this time, signal-testing experimentswere conducted to determine the appropriate run conditions for each setof measurements. Detection of free antibody was made possible using aspecies-specific, secondary antibody reagent (Goat Anti-Human IgG(H+L)-DyLight649, Part #109-495-088, Jackson ImmunoResearchLaboratories), employed at 0.75 ug/mL, 1.0 ug/mL or 2 ug/mL ininstrument buffer containing BSA at 1 mg/mL. Data obtained for eachmAb/huVEGF interaction was then simultaneously fit to a one-site bindingmodel using the vendor's software to obtain the equilibrium KDs. The KDof the affinity optimized clones were measured using KinExA and withresults summarized in Table 2.

Example 7- Assaying Affinity Optimized Variants for Binding to MurineVEGF164

Affinity optimized variants were screened to confirm binding to murineVEGF164, similarly as previously described for VEGF165 ELISA except withmurine VEGF164. EC50 values were determined using non-linear regressionanalysis (log dose response, 4-parameter fit curves) in GraphPad Prism,version 5.01 (San Diego, Calif.). Representative data are shown in FIG.6 . All affinity optimized variants exhibited strong binding to murineVEGF164, similar to the parental E06 antibody.

Example 8- Assaying Affinity Optimized Variants for Reduced Binding toVEGF121

Affinity optimized variants were screened to confirm reduced binding tohuman VEGF121 in an ELISA, similarly as previously described for VEGF165ELISA. EC50 values were determined using non-linear regression analysis(log dose response, 4-parameter fit curves) in GraphPad Prism, version5.01 (San Diego, Calif.). Representative data are shown in FIG. 7 .VEGF121 binding was reduced for J05, H1R, and H1DR in contrast to theVEGF positive control antibody with strong binding to VEGF121 (EC50 of0.0063 nM).

Example 9- Assaying Affinity Optimized Variants for Binding to VEGF189

Affinity optimized variant screening for binding to VEGF189 is in anELISA format. 96-well half well maxisorp plates will be coated with 25μl of 2 μg/mL human VEGF189 (R&D Systems), diluted in PBS without Ca++or Mg++, and refrigerated overnight. Plates will be decanted, thenblocked for 1.5 hours at 37° C. with 180 μl of Blocking Buffercontaining 3% BSA (Sigma, Cat #A-3059) and 0.1% Tween-20 in 1×PBS.Plates will be washed 3 times with 1×PBS containing 0.1% Tween-20. 50 μlof 6.7 nM and serial dilutions of affinity optimized variants, apositive control, and a negative control in blocking buffer is added induplicate and incubated at 37° C. for 1 hour. Plates will be washed 3times with wash buffer, then 50 μl of 1:5000 goat anti-human HRP IgG H+L(Jackson Immunoresearch) is added to each well and incubated at roomtemperature for 1 hour. Plates will developed by adding 50 μl of TMBsolution (KPL) to each well, followed by stopping the reaction with 50μl of 1M phosphoric acid. Plates will be read at 450 nm using amicroplate reader. Affinity optimized variants show a decrease inbinding to VEGF189.

Example 10- Functional Cell-Based Assays to Compare Potency of AffinityOptimized Variants

Human and murine pVEGFR2 cell based assays were performed as previouslydescribed to confirm potency of affinity optimized variants. EC50 valueswere determined using non-linear regression analysis (log dose response,4-parameter fit curves) in GraphPad Prism, version 5.01 (San Diego,Calif.). Representative data are shown in FIGS. 8A and 8B. Clones J05,H1R, and H1DR exhibited up to 20-fold improvement (EC50 range 2.6-10.15nM) vs. the E06 parental control (EC50=52.25 nM) in the human pVEGFR2assay (FIG. 8A), while improvement in the mouse pVEGFR2 assay was up to2-fold compared to the parental antibody (EC50 range 2.38-3.57 nM vs5.34 nM (FIG. 8B)).

Example 11- In Vivo Activity of Affinity Optimized E06 Variants

Affinity optimized variant testing for in vivo activity will be carriedout in a retinal vasculogenesis model, and a 786-0 renal cell carcinomaand B×PC3 pancreatic carcinoma model. In addition to these models,evaluation in a thrombocytopenia model will be carried out.

For the retinal vasculogenesis model, CD1 mice were intraperitoneallydosed at birth, days 1, 3, and 5. At day 8 the mice were anesthetizedand were infused with fluorescein-labeled dextran. Eyes were removed andfixed with 10% formalin before preparation of flat mounts. Flat mountswere examined by fluorescence microscopy. Neonatal retinal angiogenesisis comprised of two processes, namely, vessel migration from the opticnerve to the edge of the retina and branching. There is a decrease inbranching in the presence of H1RK compared to the untreated group.Representative data are shown in FIG. 9 . H1RK differs from H1R in thatthe light chain at position 107 (germline corrected position at 107)contains a threonine instead of a lysine.

For the 786-0 renal cell carcinoma model, 786-0 fragments will beimplanted subcutaneously into the right flank. After tumor volumereaches approximately 200 mm³, mice will be put on treatment. Mice willbe treated 2× per week for a total of 6 doses. The affinity optimizedvariants demonstrate effectiveness at reducing tumor growth, reducingtumor volume, or reducing tumor growth and tumor volume. In addition toreducing tumor growth, reducing tumor volume, or reducing tumor growthand tumor volume, the affinity optimized variants also reduce tumorvasculature. Briefly, mice will be pretreated with heparin to preventblood clotting 15 minutes prior to euthanasia. A solution of 0.1 mMsodium nitroprusside will be perfused at a rate of approximately 6mL/min. Microfil MV-122 will be prepared by mixing 8 mL of lates, 10 mLof diluent and 900 uL of cure. After the mixture settles (1 minute) itwill be perfused at a rate of approximately 2 mL/min until a totalvolume of 17 mL is administered. After 60-90 minutes the tumor will bedissected and immersed in 10% NBF for 24 hours. The sample will then betransferred to 25% ETOH/PBS, 50% ETOH/PBS, 75% ETOH/PBS, 95% ETOH, andthen 100% ETOH for 24 hours each. After the final incubation the samplewill be immersed in methyl salicylate to clear the dehydrated tumorsample before imaging by light microscopy.

For the B×PC3 pancreatic carcinoma model, female SCID mice will beimplanted subcutaneously into the right flank. After tumor volumereaches approximately 200 mm³, mice will be put on treatment. Mice willbe treated 2× per week for a total of 6 doses. The affinity optimizedvariants demonstrate effectiveness at reducing tumor growth, reducingtumor volume, or reducing tumor growth and tumor volume. In addition toreducing tumor growth, reducing tumor volume, or reducing tumor growthand tumor volume, the affinity optimized variants also reduce tumorvasculature.

For the thrombocytopenia model, a method will be used that is adoptedfrom Meyer et al, 2009 (J Thromb Haemost 7:171-81, 2009). Briefly FCgamma receptor 2A transgenic mice, 8-16 weeks old will be injected withpremixed VEGF₁₆₅, 0.6 units heparin, and an affinity optimized variantinto the lateral tail vein. Mice will then be observed for behavioralsigns of distress and scored as: (−) stopped and moved constantly fromcorner to corner, breathing normal, (+) signs of lethargy, stopped andmoved in longer duration, breathing shallow, (++) very lethargic,stopped moving, staying in mostly one side of the box, breathing deeply,(+++) sever thrombotic event-twitching and twirling, (++++) death. Theaffinity optimized variants demonstrate a reduction in thrombocytopeniaas compared to the anti-VEGF control.

Incorporation by Reference

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entireties for all purposes.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. It will beappreciated, however, that no matter how detailed the foregoing mayappear in text, the embodiments may be practiced in many ways and theclaims include any equivalents thereof.

What is claimed is:
 1. An isolated nucleic acid molecule comprisingpolynucleotides encoding an antibody comprising heavy chaincomplementarity determining regions 1-3 (HCDR1, HCDR2, and HCDR3) andlight chain complementarity determining regions 1-3 (LCDR1, LCDR2, andLCDR3), wherein HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3comprise SEQ ID NOs: 79-84, respectively.
 2. A vector comprising thenucleic acid molecule of claim
 1. 3. A cell comprising the vector ofclaim
 2. 4. A method of making an antibody comprising culturing a cellcomprising the vector of claim 2.